Team:Shenzhen/Notebook/YAO.Suicider

Context 1

• March 12th, 2012

  Events:

  March 12th. The first iGEM meeting was a very serious one, and there were only 5 members: Kang Chen Zhang Ou and Gong ,respectively.

  We decided to build a list about the whole previous iGEM projects and divided it into five parts: America for Kang and Chen, Europe for Zhang and Ou, Asia for Gong. All of us were in charge of scanning the projects, summarized them and gave the other members a brief description. Each one for one minute.

  Contributors:

  Kang Kang, Yu Chen, Junjie Zhang, Min Ou and Jianhui Gong

• March 15th – April 13th, 2012

  Events:

  Though a recruitment talk with the other classmate in BGI, nearly 20 students and two instructors became members of us. We established a team consisted of eight schools—SCUT, SCNU, SCU, UESTC, WHU, HUST, SEU, UST.

  We continued to sum up the projects from 2008 to 2011, and in the meanwhile, we separated the whole into more parts and assigned to the other members.

  Finally, we finished the summary.

  And we also discussed what we should do, we originally put up with 4 schemes: Schrodinger’s cat—randomly producing 0 and 1; Density sensing application--based on the result of JIandong Wang’s research; Christmas tree—sparks different colors ceaselessly; Yeast artificial organelle—transform mitochondria into a biofactory. The former 3 projects were gave up due to the similarity to the previous iGEM projects. We lay down our consideration on Yeast artificial organelle.

  Chen Yu search more articles about mitochondria transgene and mitochondrial signaling pathways he can and pack it as a file of endnote.

  We clarify our projects as a combination of five modules: NAD+/NADH Sensor, self-killer, post-invader, sel-control transporter and artemisinin producer.

  We divided our group into five parts according to the modules and select two members of each modules to make up two new squads—modeling construction and lab experiments.

  Contributors:

  All members

• April 14th – April 20th, 2012

  Events:

  For module of suicide:

  Originally, we simply want our yeast cell die in four different ways,two ugly: various toxicity and membrane-dissolving, two graceful: apoptosis and autophagy.

  For suicide, we put up with the idea that we let mitochondria to be the mediate between host cell death and mitochondrial dysfunction.

  Firstly, we think of self-killer through apoptosis.

  Therefore, we tried to learn about more knowledge of apoptosis in human cell and yeast cell, And figure out the difference between them by searching review and KEGG pathway.

  We know that one kind of apoptosis pathway is mediated by cytochrome C. In human cytochrome C mediated apoptosis is mainly controlled by Bcl-2 and IAP family. Bcl-2 can separate into two groups: pro-apoptosis and anti apoptosis(anti-apoptosis is consisted of two classes: BH3only and be to activate BH3).

  After we knew how cytochrome C activate the apoptosis, we tried to learn about how cytochrome C is fled away from outer mitochondrial membrane.

  Contributors:

  Yan Huang, Yang Deng, Xuefeng Luo, Senwei Tang, Jianhui Gong

• April 21st – April 27th, 2012

  Events:

  We thought about that if any materials in the apoptosis pathway or its orthology or paralogy can flee out of outer mitochondrial membrane. We found that among all the human apoptosis-related factors in cytochrome C mediated apoptosis, CytC, ENDO, PKA, Cn and ATM have the paralogy in yeast. Yeast cell has 17 chromosomes, 16 in nucleus and 1 in mitochondrion, and all paralogy mentioned above are in nucleus. [1]

  While in PE3-mediated pathway, the paralogies are ATM, CHK1, CHK2, p53R2, CytC. [2]

  Contributors:

  Ming Ni, Kang Kang, Yu Chen, Yang Deng, Jianhui Gong

• April 28th – May 4th, 2012

  Events:

  The porin on OMM can allow small molecules (<50000Dalton) in and out. Among the materials mentioned last week, cytC YJR048W has minimum aa(109), it can’t get through the pores.

  Later we try to find out the paralogy to cytC YJR048W by ncbi-blast. The paralogy in Candida glabrata( another type of yeast) has aa (104). [3]We convert 50000Da to aa number, that is 54 (for 1aa=110 Da)

  Contributors:

  Yan Huang, Xuefeng Luo, Chenran Zhou, Xiaopeng Xu, Jianhui Gong

• May 5th – May 11th, 2012

  Events:

  Try to find any peptide from mitochondrial matrix to intermembrane space or to outer membrane or even cytoplasm. But this fails. So based on the peptide sequence from mt matrix to inner membrane, we try to find out if there are any material can act like GFP when it show flurorescence, it can releast H2O2, or any material that can release H2O2, so that we can use the promoter can sense H2O2[4].

  Contributors:

  Qiwu XU Yu Chen, Kang Kang, Yan Huang , Senwei Tang, Jianhui Gong

• May 12th – May 18th, 2012

  Events:

  Figure out the function of holing to e.coli and know that holing is like bax/bak in human apoptosis pathway and therefore infer that hoin can anchor on the outer membrane of ER and mitochondrion to release the calcium and protein inside.

  Contributors:

  Deng Yan, Senwei Tang, Jianhui Gong

• May 19th – May 25th, 2012

  Events:

  Figure out that we can utilize the retrograde signaling to transfer the signal to nucleus but the premise is mt dysfunction? Maybe we can use DNase or RNase.

  Contributors:

  Kang Kang, Jianhui Gong

• May 26th – June 1st, 2012

  Events:

  How to produce material in mt? Due to the similarity between mt and prokaryotic cell. E.coli Sigma 70 promoter?

  Contributors:

  Jianhui Gong

• June 9th – June 15th, 2012

  Events:

  Brainstorming:

  1.Mt suicide itself?

      Mt autophagy or toxicity or membrane desolving.

  2.Mt mediated host cell death?

      Apoptosis

      2.1How to trigger apoptosis? Which way? CytC mediated?

          How CytC mediated apoptosis in human works? And how about in yeast cell?

          2.1.1The difference between these two?

          2.1.2Is there any paralogy can get through the inner and outer membrane of mt?

          2.1.3Is there any signal peptide from mt matrix to IM, OMM, cytoplasm?

          2.1.4There is retrograde signaling but the premise is the mt respiration hardship.

          2.1.5Respiration hardship can be imitated by mt dysfunction such as DNA fragmentation.

          2.1.6How to make DNA fragmentation?

              DNase? RNase? Phosphodiesterase?

          2.1.7Any repressor to TCA cycle?

      2.2How to sense the signal given to mt?

          2.2.1For the same reason that the inner membrane of mt is extremely close, and its cardiolipin resist to the polar material

          2.2.2Sense the signal from biosensor? Biosensor is not completed.

          2.2.3So we use T7 RNAP/T7 promoter to mimic the signal given to mt.

      2.3How to release CytC?

          2.3.1Bax/Bak?

          2.3.2Holin??

      2.4How nucleus sense signal and produce holin?

          2.4.1Retrograde signaling RTG pathway from mt to nucleus. [5]

  Contributors:

  Jianhui Gong

• June 16th – June 22nd, 2012

  Events:

  Design the verification experiment for the whole story.

• 1. Originally, we try to construct such a artificial organell YAO that YAO receives signal either from biosensor (internal)or from cytoplasm (external) or consequently trigger the host cell apoptosis. However, for YAO are not completed, we use mitochondrion as experiment target. And since we can’t rely on the availability of YAO. sensor, the idea that Initial signal is given to YAO’s promoter is replaced by that nucleus produce T7 RNAP and transport it into mitochondrion to activate the T7 promoter inside it.

• 2. The final story is consist of six parts:

•     a. Signal imitation: After galactose is added to the medium, RNAP can bind to Gal promoter and start transcribe and produce T7 RNAP with signal peptide to mitochondrial matrix. Then T7 RNAP get into mitochondrial matrix.

•     b. Mitochondrion dysfunction: Once T7 promoter works, DNase is produced to fragmentate the mtDNA which can lead to Respiration difficulty.

•     c. Retrograde signaling from mitochondrion: Mitochondrial dysfunction can trigger the RTG pathway which is the only known signaling from mt to nucleus.

•     d. Holin production: DLD3 promoter is the only stable downstream promoter relevant with RTG pathway. It can be binded and activated by RTG1/3p which is final production of RTG pathway. Once mitochondrial dysfunction exits, holin with DLD3promoter can be expressed.

•     e. Holin transformation and destruction: Once holin is produced, it can anchor on the outer membrane of mitochondria and ER, and form dimer with each other. Further more, they can form into a multidimer called dead draft if holin reachs a certain concentration. Dead drafts work like a large pore and release the apoptosis factor: cytochrome C, aif1 and calcium into cytoplasm.

•     f. Apoptosis of host: Apoptosis factor cytochrome C and largely fleeing calcium can trigger the caspase-like pathway. Aif1 such as endo G can.get into nucleus and fragmentate DNA. Finally, host cell is suppressed or even die.

• Contributors:

  Qiwu Xu, Jianhui Gong

• June 23rd – June 29th, 2012

  Events:

  For T7 imitation system:

•     1. Verify the function of T7 RNAP/Promoter system in cytoplasm. If it works, fluorescence of GFP can be detected in cytoplasm.

•     2. Verify the function of T7 RNAP/Promoter system in mitochondrion or unfinished YAO, basing on the availability of signal peptide from cytoplasm to mitochondrial matrix. If it works, fluorescence of mtGFP can be detected in mitochondrion.

• For retrograde signaling part:

•     3. Because DLD3 is one of the target of RTG pathway of yeast, lowering the basal expression is required. Verify the basal expression of gene downstream DLD3 promoter. Roughly judge the brightness of GFP, and consequently select a suitable degradation tag for gene downstream DLD3 promotor.

•     4. Verify the function of selected degradation tag by using UV as a imitation of DNase produced in mitochondrion that can lead to mitochondrion dysfunction which will drive the gene expression downstream DLD3 promoter through Retrograde signaling from mitochondrion to nucleus.

•     5. Verify that holing can make pores on the inner membrane of mitochondrion. This is the alterant of DNase causing mt dysfunction. And it can also produce a membrane without mtDNA.

• For host cell suicide part:

•     6. Verify the function of holing in yeast to prove that holing can act as bax/bak protein in human cell as a apoptosis activator. If it works, we can see that between the engineered and versus one, the growthrate curve (OD600) of engineered one tend to be lower than that of versus one.

• Contributors:

  Qiwu Xu, Yang Deng, Jianhui Gong 

  1.<a href="http://www.genome.jp/kegg-bin/show_pathway?map04210">http://www.genome.jp/kegg-bin/show_pathway?map04210</a>

  2.       <A href="http://www.genome.jp/kegg-bin/show_pathway?hsa04115">http://www.genome.jp/kegg-bin/show_pathway?hsa04115</A><o:p></o:p>

  3.       <A href="http://www.genome.jp/dbget-bin/www_bget?cgr:CAGL0I01408g">http://www.genome.jp/dbget-bin/www_bget?cgr:CAGL0I01408g</A>

• 4.   Prindle, A. et al. A sensing array of radically coupled genetic ‘biopixels’. Nature. 2011;481:39-44.

• 5.    Liu, Z. Butow, R. A. Mitochondrial retrograde signaling. Annu Rev Genet. 2006;40:159-85.

• 6.    Shi, Y. Sun, J.Current advance in the topological structure and function of holin encoded by bacteriophage lambda. Wei Sheng Wu Xue Bao. 2012 ;52:141-5.

• 7.    Agu, C. A et al. Bacteriophage-encoded toxins: the lambda-holin protein causes caspase-independent non-apoptotic cell death of eukaryotic cells. Cell Microbiol. 2007:1753. Lipton, S. A.
  

• 8.    Bossy-Wetzel, E. Dueling activities of AIF in cell death versus survival: DNA binding and redox activity. Cell. 2002;111:147-50.

• 9.    Buttner, S. Endonuclease G regulates budding yeast life and death. Mol Cell. 2007 ;25:233-46.

• 10.  Spencer, S. L. Sorger, P. K. Measuring and modeling apoptosis in single cells. Cell. 2011;144:926-39.

• 11.  Li, P. Nijhawan, D. Wang, X. Mitochondrial activation of apoptosis. Cell. 2004;116:S57-9.

• 12.  Pinkham, J. L. Dudley, A. M. Mason, T. L. T7 RNA polymerase-dependent expression of COXII in yeast mitochondria. Mol Cell Biol. 1994 ;14:4643-52.

Context 2